Influenza vaccine production in liquid cell culture

ABSTRACT

Infectivity and replication of influenza viruses in successive numbers of cells of the same liquid cell culture is assured by including a protein hydrolyzing enzyme in the culture during virus incubation. Technique overcomes &#34;one-step growth cycle&#34; of virus and allows commercial influenza vaccine production from liquid cell cultures instead of from more costly embryonated chicken eggs. Resulting vaccine is thus substantially free of egg proteins.

This application is a continuation-in-part application of Ser. No.039,236, filed May 15, 1979, abandoned.

BACKGROUND OF THE INVENTION

1. Field

This disclosure is concerned generally with a novel influenza viruspropagation medium and specifically with the use of that medium forinfluenza vaccine production.

2. Prior Art

Influenza vaccines have been in use since the early 1940's for humanvaccination and since the late 1960's for equine vaccination. Allinfluenza vaccines presently used are made by growing the vaccine virusstrains in embryonated chicken eggs. The resulting virus strains arethen used for making live virus vaccines or further processed to makekilled virus vaccines.

It is generally known by virologists that influenza viruses grow to avery limited degree in cell cultures. The growth is referred to as a"one-step growth cycle"; that is, only the originally infected cellsreplicate viruses. This phenomenon is described, for example, by Daviset al., MICROBIOLOGY, Harper and Row Publishers, Chapter 44, pp. 1138-39(1968). Since the viruses of the originally infected cells are unable toinfect successive numbers of cells in the same cell culture, theresulting yields are far too low to be useful in the preparation ofvirus vaccines. Thus, liquid cell cultures have not been used forcommercial production of influenza virus vaccines.

Embryonated chicken eggs are used to produce viruses with titerssufficiently high enough to be useful in the preparation of vaccines.Unfortunately, chick embryo-grown viruses usually require concentration,and, in the case of human vaccines, also require some form ofpurification to reduce toxic reactions due to the undesirable eggproteins. The use of the eggs for vaccine production is time consuming,labor intensive, requires relatively high material costs, and the yieldfrom one egg is commonly only enough to produce vaccine for about one to1.5 doses. Thus, the manufacture of millions of doses requiresinnoculating and harvesting millions of embryonated eggs.

Recently, it has been noted that a wide variety of influenza A virusescomprising human, equine, porcine, and avian strains, grew productivelyin an established line of canine kidney cells under an overlay mediumcontaining trypsin and formed well-defined plaques regardless of theirprior passage history. See the article by K. Tobita et al., "PlaqueAssay and Primary Isolation of Influenza A Viruses in an EstablishedLine of Canine Kidney Cells (MDCK) in the present of Trypsin", Med.Microbiol. Immunol. 162, 9-14 (1975). See also the article byHans-Dieter Klenk et al., "Activation of Influenza A Viruses by TrypsinTreatment", Virology 68, 426-439 (1975). It should be noted that in theabove reports the effects of trypsin on influenza virus propagation wereobserved in semi-solid cultures in plaque formation assays and isolationtechniques, neither of which are concerned with liquid cell cultures orthe large scale or commercial propagation of influenza viruses forvaccine production. The term liquid cell culture used herein describesthe in vitro growth of cells and propagation of virus in a chemicallydefined liquid medium.

Quite surprisingly, we have now found that proteolytic enzymes can alsobe used in liquid cell cultures to facilitate infection of successivenumbers of cells in the same cell culture. By thus overcoming thelimitations of the "one-step growth cycle" of past liquid cell culturetechniques, it is possible to achieve an influenza virus yield which isin the range of about 1,000 to 10,000 fold greater than non-proteasetreated cultures. This makes feasible the use of liquid cell culturingtechniques for the commercial production of influenza vaccines, therebyavoiding the disadvantages associated with using embryonated chickeneggs. Details of our culturing medium, virus propagation techniques, andvaccine production and use methods are disclosed herein.

SUMMARY OF THE INVENTION

Our influenza virus propagation medium comprises a cell culture capableof being infected with an influenza virus, an influenza virus, and aprotein-hydrolyzing enzyme, the amount of enzyme being sufficient toovercome the one-step growth cycle of the virus. Our virus propagationtechnique comprises the steps of inoculating or infecting a liquidinfluenza virus cell culture with the influenza viruses, incubating theinoculate in the presence of a protein-hydrolyzing enzyme underconditions sufficient to assure maximum virus growth (or maximumcytopathic effect) and harvesting the virus. Infection of the cells withthe virus may occur before or after cell monolayer formation or,alternatively, by simply infecting a liquid suspension of the cells. Ourvaccine production method includes the subsequent step of killing theharvested virus or attenuating by further cell culture passage forvaccine use. Especially preferred embodiments involve the use of theprotease trypsin in conjunction with a dog kidney cell line to propagateany of several types of influenza viruses.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-5 are bar graphs illustrating the increases in titer achievedwhen the indicated virus strains were incubated in a liquid cell culturein the presence of various amounts of a typical protease, trypsin.Results are reported as geometric mean titers.

SPECIFIC EMBODIMENTS

The influenza virus and vaccine production techniques of this disclosurecontemplate the use of influenza vaccine viruses. As used herein, theterm influenza virus includes any viruses capable of causing a febrilediease state in animals (including man) marked by respiratory symptoms,inflamation of mucous membranes and often systemic involvement. Themedium and methods of this disclosure are especially useful in theproduction of a variety of influenza viruses, including human, equine,porcine, and avian strains. Examples of the production of typical Type Aand B Influenza viruses are described below.

The vaccine preparations contemplates by this disclosure include anypreparation of killed, living attenuated, or living fully virulentinfluenza viruses that can be administered to produce or artificiallyincrease immunity to any influenza disease. In preferred embodiments,the vaccine comprises an aqueous suspension of the virus particles inready to use form.

The cell cultures contemplated as being useful in carrying out theprinciples of this disclosure include any animal cell line or cellstrain capable of being infected by, and which allows the replicationof, one or more given influenza virus strains. Although a number of suchcells are known and thought to be useful for the techniques disclosedherein, we have had especially good results with an established cellline known as the Cutter Laboratories Dog Kidney (CLDK) cell line. TheCLDK cell line has been approved by the U.S. Department of Agriculturefor use in producing veterinary vaccines and is similar to the madinDarby Dog Kidney Cell Line (ATCC No. CCL 34) and to the dog kidney cellline described in U.S. Pat. No. 3,616,203 to A. Brown. A brief historyand description of the specific master cell stock used for the cell lineof the Examples follows although it should be understood that thetechniques disclosed herein are thought to be useful with any influenzavirus susceptible cell culture.

CUTTER LABORATORIES DOG KIDNEY (CLDK) CELL LINE HISTORY

The parent line of CLDK was initiated and established at CutterLaboratories, Inc., Berkeley, Calif. from the kidney of an apparentlynormal beagle dog obtained from the University of California at Davis.The line was maintained on 0.5% lactalbumin hydrolysate and 0.2% yeastextract in Earle's balanced salt solution plus 5% calf, lamb, or horseserum and antibiotics, cultivated by the methods of J. S. Younger (Proc.of Exp. Biol., and Med., v 85 202).

A frozen ampoule of the 142nd passage of this cell line was subsequentlyplanted in a 75 cm² (250 ml) falcon flask, in tissue culture mediumconsisting of Earle's balanced salt solution Minimum Essential Medium(MEM) and 10% fetal bovine serum. The cells were subcultured in the samemanner and medium to prepare the frozen Master cell stock at passage148.

An ampoule of the Master cell stock was thawed, planted, and seriallysubcultured 20 times to obtain bottle cultures of the 168th passage.These cells were then frozen.

DESCRIPTION OF MASTER CELL STOCK (MSC)

Number of Serial Subcultures from Tissue of Origin: 148

Freeze Medium: Minimum essential medium (Eagle) in Earle's BSS withreduced bicarbonate (1.65 gm/L) 80%; fetal bovine serum 10%; dimethylsulfoxide 10%.

Viability: Approximately 75% (dye exclusion).

Culture Medium: Minimum essential medium (Eagle) in Earle's BSS withreduced bicarbonate (1.65 gm/L) 90%; fetal bovine serum 2-10%;antibiotic penicillin and streptomycin 100 U. or γ/ml.

Growth Characteristics of Thawed Cells: An inoculum of 3×10⁶ viablecells/ml cultured in the above culture medium at 37° C. in a closedsystem, multiplies approximately 6-8 fold in 5 days.

Morphology: Epithelial-like.

Karyology: Chromosome Frequency Distribution 100 Cells: 2N=72.

    ______________________________________                                        Cells    1       1     2     4   6     9   69  5   3                          Chromosomes                                                                            60      65    68    69  70    71  72  73  74                         ______________________________________                                    

No marker chromosomes.

Sterility Tests: Free of Mycoplasma, bacteria and fungi.

Species: Confirmed as canine by immunofluorescence test.

Virus Susceptibility: Susceptible to influenza viruses, rabies virus,infectious bovine rhinotrachetis, infectious canine hepatitis, caninedistemper virus, and possibly other viruses.

The protein hydrolyzing enzymes (proteolytic enzyme or protease)contemplated as being useful for purposes of this disclosure includewell known proteases such as trypsin, chymotrypsin, pepsin, pancreatin,papain, pronase, carboxypeptidase, and the like, with trypsin being anespecially preferred enzyme. The exact mechanism(s) by which a proteasesuch as trypsin enhances the influenza virus infectivity is not fullyknown. One possible mechanism has been suggested in the above citedarticle by Klenk et al.

As described below, the amount of active protease required to enhancesuccessive infectivity should be at least enough to overcome thelimitations of the one-step growth cycle but, in the case of confluentmonolayer cultures, not so much as to cause a sloughing of confluentcells from the surfaces of the tissue cultivation vessel (i.e. the innersurface of a roller bottle). In the case of the specific enzyme used inthe examples below, we prefer that the amount of the enzyme be in therange of about 4 to 25 micrograms per ml (μg/ml) of liquid tissueculture medium, preferably about 10 μg/ml.

VIRUS PROPAGATION METHODS

The influenza virus propagation method of this disclosure comprises thethree general steps of infecting a portion of the cells of a liquid cellculture with the influenza virus, incubating the cells in the presenceof a proteolytic enzyme under conditions sufficient to assure maximumcytopathic (CP) effect, and harvesting the viruses from the culture.Vaccine preparation comprises the subsequent step of modifying theharvested virus by known techniques to result in a live virulent,attenuated, or killed (inactive) vaccine preparation. The vaccine may beavailable in dry form, to be mixed with a diluent, or in liquid, readyto use form. Suitable adjuvants may be included, as described below, toenhance immunogenicity.

The protease may be added to an aqueous suspension cell culture orbefore or after formation of a confluent monolayer culture. Examples ofsome of the various propagation methods are described below.

METHOD A-Infection of Pre-formed Monolayers

1. Cells are grown to confluency in culture containers such as rollerbottles, Povitsky flasks, or Roux bottles utilizing cell culture growthmedia known to the art.

2. Prior to infection, the growth medium is removed from the cellmonolayers.

3. The influenza virus working seed is diluted in growth mediumcontaining additional vitamins, non-essential amino acids, L-glutamine,dextrose, and antibiotics with the pH adjusted to 6.6-6.8.

4. A quantity of the diluent containing virus is added to the cellmonolayer in quantities ranging from 10% to 100% of the final harvestvolume.

5. The infected monolayers are incubated at 34°-37° C. for 1 to 72hours.

6. Protease, alone or in combination with the virus propagation media,is added at a concentration which will stimulate multiple cycle growthwithout producing cell slough. For trypsin, this optimum concentrationis between about 8 and 15 μg/ml.

7. The virus growth containers are once again incubated at 34°-37° C.until maximum cytopathic effects are observed. At this point the virusfluids are harvested.

8. Harvest involves shaking the containers vigorously to remove cellsand transferring fluids and cells to a sterile container for furtherprocessing.

METHOD B-Infection of Liquid Cell Suspension Prior to MonolayerFormation

1. Cells are removed from growth containers using conventionalprocedures.

2. Cells are concentrated by centrifugation, then resuspended in aquantity of fresh growth medium containing additional vitamins,non-essential amino acids, L-glutamine, dextrose, and antibiotics.

3. Influenza virus is then added to this concentrated cell suspension.

4. The cell virus suspension is incubated (25° C.-37° C.) in a sterile,closed container (such as a screwcap Ehrlenmeyer flask) while beingmixed on a magnetic type stirrer or rotary shaker for 10 minutes to 4hours.

5. Aliquots of the cell virus suspension are placed into growthcontainers (roller bottles, Roux bottles, Povitsky flasks) with the fullvolume of media containing ingredients indicated in B-2 plus 5% fetalcalf serum.

6. Growth containers are incubated at 34°-37° C. until confluentmonolayers are formed (approximately 2-4 days).

7. After monolayers have formed, protease is added at a concentrationwhich will stimulate multiple cycle growth without producing cellslough. For trypsin, this optimum concentration is between 10 and 25μg/ml.

8. Growth containers are incubated at 34°-37° C. until maximumcytopathic effects are observed. Virus is then harvested.

9. Harvest involves shaking containers vigorously to remove cells andtransferring cells and fluids to a sterile container for furtherprocessing.

METHOD C-Infection of Liquid Suspension Culture

1. Cells adapted to suspension culture are grown to an optimum count ingrowth medium in suspension growth containers.

2. Cells are centrifuged and resuspended in a quantity of fresh mediumcontaining additional vitamins, non-essential amino acids, L-glutamine,dextrose, and antibiotics.

3. Influenza virus is then added and the culture is incubated at 34°-37°C. for 10 minutes to several hours.

4. Fetal calf serum may be added and the culture suspension is furtherincubated at 34°-37° C. for 1 to 72 hours.

5. Protease is added at a concentration which will allow multiple cyclegrowth without producing a detrimental effect on the cells. For trypsin,the optimum concentration is between 4 and 25 μg/ml.

6. Incubation at 34°-37° C. is continued until maximum cytopathiceffects are observed at which time fluids are harvested.

7. Harvest involves transfer of fluids to a sterile container forfurther processing.

Specific examples of the use of our techniques and media for thepropagation of selected strains of influenza viruses follow. Unlessotherwise indicated, we used conventional tissue culturing techniquesknown to the art. Since, both the cell and medium preparation techniquesare well known, they are not described here in detail.

EXAMPLE 1

The A2 Equine Influenza Virus, designated Miami strain, was originallyisolated from a horse at the University of Miami. This virus wasobtained from the University of Pennsylvania Medical School where sixpassages were made in chick embryo. The seventh passage was made at andobtained from Lederle Laboratories. The strain underwent further chickembryo passage and was used at passages 11-16.

The present preferred method of tissue culture propagation of the A2strain involves infection of a young, confluent monolayer of CLDK cells.Cells are planted in roller bottles, using Hank's Minimum EssentialMedium (MEMH) containing the following ingredients:

Fetal Bovine Serum, 5-10%

Non-Essential Amino Acids, 10 ml/l (Gibco)

L-Glutamine, 10 ml/l (Gibco)

Neomycin Sulfate, 30,000 mcg/l

Polymyxin B, 30,000 units/l

Mycostatin, 25,000 units/l

The cells are usually confluent with 72 hours at which time the mediumis poured off and the cells are infected. The inoculating mediumcontains A2 virus diluted to an Egg Infective Dose₅₀ (EID₅₀) titer ofapproximately 10³.0 /ml in MEMH supplemented with the followingingredients:

50% Dextrose, 2.6 ml/l

MEM Vitamins, 30 ml/l (Gibco)

Non-Essential Amino Acids, 10 ml/l (Gibco)

L-Glutamine, 10 ml/l (Gibco)

Neomycin Sulfate, 30,000 mcg./l

Polymyxin B, 30,000 units/l

Mycostatin, 25,000 units/l

Inoculating medium equivalent to 14% of the final volume is added toeach roller bottle and the containers are incubated at 34°-35° C. for 72hours. At this time, the remaining medium (86%) containing 12 μg/ml ofsterile trypsin (Sigma, 1:250) solution (0.1 g/100 ml), is added to eachroller bottle. Containers are again incubated at 34°-35° C. untilmaximum cytopathic effect is observed (48-72hours) at which time thefluids are harvested. Harvest involves vigorously shaking each rollerbottle to remove any attached cells and transferring cells and virusfluids to a sterile batching container for further processing.

Harvest EID₅₀ titers of A2 Influenza Virus grown using this techniqueare shown in Table 1. Also, see FIG. 1. A comparison is made with A2virus grown by the same method but without adding trypsin.

                  TABLE 1                                                         ______________________________________                                        EID.sub.50 OF                                                                 MIAMI STRAIN EQUINE INFLUENZA VIRUS                                           GROWN IN CLDK CELLS WITH AND WITHOUT TRYPSIN                                  Amt. Trypsin                                                                           Titer (EID.sub.50 /ml)                                                                           Fold Increase                                     (μg/ml)                                                                             Input     Harvest  With Trypsin                                      ______________________________________                                        None     10.sup.2.6                                                                              10.sup.6.2                                                                             --                                                None     10.sup.3.2                                                                              10.sup.6.2                                                                             --                                                5        10.sup.2.9                                                                              10.sup.8.3                                                                             126                                               5        10.sup.2.9                                                                              10.sup.8.1                                                                             79                                                5        10.sup.2.6                                                                              >10.sup.9.2                                                                            >1.000                                            10       10.sup.2.9                                                                              10.sup.9.2                                                                             1.000                                             10       10.sup.2.9                                                                              10.sup.8.5                                                                             200                                               10       10.sup.3.2                                                                              10.sup.8.2                                                                             100                                               10       10.sup.3.2                                                                              .sup. 10.sup.10.0                                                                      6.310                                             10       10.sup.3.1                                                                              .sup. 10.sup.14.5                                                                      199,526.232                                       15       10.sup.3.1                                                                              10.sup.8.9                                                                             501                                               ______________________________________                                    

Incorporation of trypsin into the growth medium produced a geometricmean increase of 3.2 logs or 1711 times as many virus particles/mlduring production of the Miami strain.

EXAMPLE 2

A sample of virulent type A1 Equine Influenza virus was obtained fromthe University of Pennsylvania Medical School. The strain, designatedPennsylvania (A1), has been isolated from a horse and passaged in chickembryo six times. The strain has undergone further chick embryo passageand is being used for tissue culture production at passages 12-17.

The preferred method of tissue culture propagation of the A1 straininvolves infection of a suspension of CLDK cells prior to monolayerformation. CLDK cells at a concentration of approximately 10⁵.5 /ml,Pennsylvania strain of virus at an EID₅₀ titer of 10³.0 /ml to 10⁵.0/ml, and Hank's Minimum Essential Medium (MEMH) supplemented with theingredients listed below are incubated at 25° C. while being mixed on amagnetic stirrer in a closed, sterile Ehrlenmeyer flask. The pH ismaintained at 6.7-6.8 with I N HCI during the 2-3 hour incubationperiod.

Supplemented MEMH

50% Dextrose, 2.6 ml/l

MEM Vitamins, 30 ml/l (Gibco)

Non-Essential Amino Acids, 10 ml/l (Gibco)

L-Glutamine, 10 ml/l (Gibco)

Neomycin Sulfate, 30,000 meg./l

Polymyxin B, 30,000 units/l

Mycostatin, 25,000 units/l

After this suspension incubation, 10 ml aliquots of the cell virussuspension are added to roller bottles containing 1 liter of MEMHsupplemented as listed above and containing 5% Fetal Calf Serum. Theroller bottles are incubated at 34°-35° C. until the monolayer isconfluent (approximately 48-72 hours) after which 20 ml of a sterile 1mg/ml trypsin solution (Sigma 1:250) is added to each roller bottle. Theroller bottles are again incubated at 34°-35° C. until the maximumcytopathic effect is observed (3-5 days). The virus fluids are harvestedby vigorously shaking each roller bottle to remove cells which remainattached and transferring cells and fluids to a sterile container forfurther processing.

Harvest EID₅₀ titers of the A1 Influenza virus grown using thistechnique are shown in Table 2. Also, see FIG. 2. A comparison is madewith A1 virus grown by the same method excluding trypsin

                  TABLE 2                                                         ______________________________________                                        EID.sub.50 TITERS OF                                                          PENNSYLVANIA STRAIN EQUINE INFLUENZA VIRUS                                    GROWN IN CLDK CELLS WITH AND WITHOUT TRYPSIN                                  Amt. Trypsin                                                                             Titer (EID.sub.50 /ml)                                                                        Fold Increase                                      (μg/ml) Input      Harvest  With Trypsin                                   ______________________________________                                        None       10.sup.5.3 10.sup.5.9                                                                             --                                             None       10.sup.4.9 10.sup.5.4                                                                             --                                             10         10.sup.5.3 10.sup.7.4                                                                             50                                             10         10.sup.4.9 10.sup.6.8                                                                             13                                             10         10.sup.5.1 10.sup.8.0                                                                             200                                            20         10.sup.5.3 10.sup.8.7                                                                             1.000                                          20         10.sup.4.9 10.sup.8.5                                                                             631                                            20         10.sup.5.0 10.sup.7.1                                                                             25                                             20         10.sup.4.9 10.sup.8.3                                                                             398                                            20         10.sup.3.2 10.sup.9.2                                                                             3.162                                          20         10.sup.3.2 10.sup.7.5                                                                             63                                             20         10.sup.3.2 10.sup.8.2                                                                             316                                            ______________________________________                                    

Incorporation of trypsin into the growth medium produced a geometricmean increase of 2.3 logs or 187 times as many virus particles/ml duringproduction of the Pennsylvania strain.

EXAMPLE 3

A strain of Human Influenza virus designated B/Hong Kong/5/72 (BX-1) wasreceived from The Center for Disease Control in Atlanta, Ga. This waspassaged once in embryonated chicken eggs and frozen at -70° C. asworking seed virus.

The preferred method of tissue culture propagation of the B/HongKong/5/72 strain involves infection of a young confluent monolayer ofCLDK cells similar to that in Example 1. Cells are grown as described inExample 1. Growth medium is removed from cells and discarded. Cells arethen infected with virus diluted in the inoculating medium listed inExample 1 to an EID₅₀ titer of approximately 10³.0-5.0 /ml. The inoculumconsists of a volume equivalent to 33.3% of the final harvest volume.Containers are incubated at at 34°-35° C. for 40-48 hours after whichthe remaining medium (66.7%) containing 12 μg/ml of trypsin solution(0.1 g/100 ml) is added to each container. Incubation at 34°-35° C. iscontinued until the maximum cytopathic effect is observed (48-72 hours)at which time the virus fluids are harvested. Harvest involvesvigorously shaking each container to remove cells and transferring cellsand fluids to a sterile container for further processing.

Harvest EID₅₀ titers of B/Hong Kong/5/72 Influenza virus grown usingthis technique are shown in Table 3. Also, see FIG. 3. A comparison ismade with this strain grown by the same method but without addingtrypsin.

                  TABLE 3                                                         ______________________________________                                        EID.sub.50 TITERS OF B/HONG KONG/5/72 STRAIN                                  HUMAN INFLUENZA VIRUS GROWN IN                                                CLDK CELLS WITH AND WITHOUT TRYPSIN                                           Amt. Trypsin                                                                              Titer (EID.sub.50 /ml)                                                                            Fold Increase                                 (μg/ml)  Input   Harvest     With Trypsin                                  ______________________________________                                        None        10.sup.3.2                                                                            10.sup.6.0  --                                            None        10.sup.5.7                                                                            10.sup.6.4  --                                            8           10.sup.3.0                                                                            10.sup.8.7  316                                           8           10.sup.2.0                                                                            10.sup.9.0  631                                           8           10.sup.4.5                                                                            10.sup.9.5  1.995                                         8           10.sup.3.5                                                                            .sup. >10.sup.10.2                                                                        >10.000                                       ______________________________________                                    

Incorporation of trypsin into the growth medium produces a geometricmean increase of 3.1 logs or 1412 times as many virus particles duringproduction of the B/Hong Kong strain.

EXAMPLE 4

A strain of Human Influenza virus designated A/Texas/1/77 was receivedfrom The Center for Disease Control in Atlanta, Ga. This was passagedonce in embryonated chicken eggs and frozen at -70° C. as working seedvirus.

The preferred method of tissue culture propagation of the A/Texas/1/77strain is that described in total in Example 3.

Harvest EID₅₀ titers of A/Texas/1/77 Human Influenza virus grown usingthis technique are shown in Table 4. Also, see FIG. 4. A comparison ismade with this strain grown by the same method but without addingtrypsin.

                  TABLE 4                                                         ______________________________________                                        EID.sub.50 TITERS OF A/TEXAS/1/77 STRAIN                                      HUMAN INFLUENZA VIRUS GROWN IN                                                CLDK CELLS WITH AND WITHOUT TRYPSIN                                           Amt. Trypsin                                                                           Titer (EID.sub.50 /ml)                                                                            Fold Increase                                    (μg/ml)                                                                             Input     Harvest   With Trypsin                                     ______________________________________                                        None     --        10.sup.5.2                                                                              --                                                8       10.sup.5.1                                                                              10.sup.8.9                                                                              5.012                                             8       10.sup.4.1                                                                              .sup. >10.sup.10.2                                                                      >100.000                                         10       10.sup.3.0                                                                              10.sup.8.3                                                                              1.259                                            10       10.sup.2.0                                                                              10.sup.9.8                                                                              39.811                                           ______________________________________                                    

Incorporation of trypsin with the growth medium produced a geometricmean increase of 4.1 logs or 12590 times as many virus particles/mlduring production of the A/Texas strain.

EXAMPLE 5

A strain of Human Influenza virus designated A/USSR/90/77 was receivedfrom the Center for Disease Control in Atlanta, Ga. This was passagedonce in embryonated chicken eggs and frozen at -70° C. as working seedvirus.

The preferred method of tissue culture propagation of the A/USSR/90/77strain is that described in total in Example 3.

Harvest EID₅₀ titers of A/USSR/90/77 Human Influenza virus grown usingthis technique are shown in Table 5. Also, see FIG. 5. A comparison ismade with this strain grown by the sane method but without addingtrypsin.

                  TABLE 5                                                         ______________________________________                                        EID.sub.50 TITERS OF A/USSR/90/77 STRAIN                                      HUMAN INFLUENZA VIRUS GROWN IN                                                CLDK CELLS WITH AND WITHOUT TRYPSIN                                           Amt. Trypsin                                                                             Titer (EID.sub.50 /ml)                                                                            Fold Increase                                  (μg/ml) Input      Harvest  With Trypsin                                   ______________________________________                                        None       10.sup.4.0 10.sup.6.3                                                                             --                                             10         10.sup.4.0 10.sup.8.7                                                                             251                                            10         10.sup.4.0 10.sup.9.0                                                                             501                                            10         10.sup.4.5 10.sup.8.4                                                                             126                                            ______________________________________                                    

Incorporation of trypsin into the growth medium produced a geometricmean increase of 2.4 logs or 251 times as many virus particles/ml duringproduction of the A/USSR strain. VACCINE PREPARATION

EXAMPLE 6-VIRUS ATTENUATION

Attenuation of the virus from harvested fluids of Example 1 isaccomplished chemically or by standard serial passages includingterminal dilution passage techniques wherein a sufficient number ofpassages in a susceptible cell culture is employed until the virus isrendered non-pathogenic without loss of immunogenicity. A vaccineprepared in these manners will stimulate an immune response in animalssusceptible to disease without producing the clinical sysmptoms normallydue to the virulent agent to any significant degree. The propagation canbe done in the same or different tissues as those employed in thepreceding passage.

EXAMPLE 7-VIRUS INACTIVATION

The technique is similar to that described in Examples 1 and 2 but theharvest viral ledan fluids are further processed by inactivation with0.1% concentration of formaldehyde (range 0.05 to 0.2%) and the treatedmaterial is incubated at 4° C. for 10 to 14 days. Testing of the finalinactivated viral preparation showed it to be free from live virus.Adjuvants known to the art, such as aluminum hydroxide, alum, aluminumphosphate, Freund's, or those described in U.S. Pat. Nos. 3,790,665 and3,919,411 may be added. The preferred adjuvant of this disclosure andthat used in our vaccine is an acrylic acid polymer crosslinked with apolyallylsaccharide (Carbopol 934 P) similar to that described in theabove patents.

EXAMPLE 8-INACTIVATED VIRUS VACCINE PREPARATION AND USE

A 1.0 ml equine dose consists of 0.45 ml of the Pennsylvania (A1)strain, 0.45 ml of the Miami (A2) strain and 0.10 ml of the Carbopoladjuvant. Equal parts of the inactivated vaccine strains obtained fromExample 7 were mixed and 1 ml aliquots administered to 19 horses by theintramuscular route. No clinical disease or symptoms of influenza werenoted in any of the horses after vaccination. Antibody titers againstboth Equine Influenza A1 and Equine Influenza A2 were obtained on bloodsera of all animals at 2, 4, and 8 weeks following inoculation. Theseare compared to the pre-inoculation levels (Table 6) using the standardhaemagglutination inhibition test (DIAGNOSTIC PROCEDURES for Viral andRickettsial Infections, Fourth Edition; Lenette and Schmidt, pp. 665-66(1969). American Public Health Association, New York, N.Y. 10019).

                  TABLE 6                                                         ______________________________________                                        ANTIBODY RESPONSES                                                            (HAEMAGGLUTINATION INHIBITION)                                                       Equine Influenza A1                                                                             Equine Influenza A2                                                  2      4    8         2    4    8                             Horse No.                                                                              Pre.   wk.    wk.* wk.  Pre. wk.  wk.* wk.                           ______________________________________                                        2        <8     8.192  8.192                                                                              8.192                                                                              <8   512  128  256                           11       <8     128    256  128  <8   64   1.024                                                                              64                            13       <8     128    256  2.048                                                                              <8   256  256  1.024                         19       <8     256    64   64   <8   128  128  64                            21       <8     128    64   512  <8   64   64   512                           23       <8     256    128  128  <8   512  512  128                           29       <8     128    32   1.024                                                                              <8   256  64   128                           32       <8     1.024  256  2.048                                                                              <8   64   64   128                           37       <8     128    32   512  <8   64   32   64                            38       <8     1.024  512  1.024                                                                              <8   512  256  64                            40       <8     256    128  1.024                                                                              <8   128  128  128                           55       <8     512    512  8.192                                                                              <8   512  256  256                           61       <8     512    64   256  <8   1.024                                                                              128  128                           68       <8     128    32   128  <8   512  256  256                           79       <8     64     128  2.048                                                                              <8   256  128  128                           121      <8     <8     32   512  <8   1.024                                                                              256  64                            125      <8     256    32   128  <8   256  128  128                           128      <8     512    128  512  <8   256  128  256                           129      <8     256    128  2.048                                                                              <8   512  512  256                           ______________________________________                                         *Day of booster                                                          

As can be seen from Table 6, antibody developed to both viral antigensin horses receiving the inoculations. These vaccinates would be immuneto Equine Influenza since antibody titers in excess of 1:20 to A2 and1:60 for A1 are accepted as being protective by the National VeterinaryServices Laboratories of the U.S. Department of Agriculture.

Clinical trials in 420 horses of various breeds and ages showed thevaccine to be safe and to produce no untoward reactions afterintramuscular inoculation.

EXAMPLE 9-ATTENUATED VIRUS VACCINE USE

Three horses were given 5 ml of the live, attenuated Equine Influenza A2vaccine strain of Example 6 by the intranasal route. No clinical diseaseor symptoms of influenza were noted in any of the horses aftervaccination. Antibody titers against the vaccine strain were obtained onblood sera of all animals at 1, 2, and 4 weeks following inoculation andcompared to the pre-inoculation level using the standardhaemagglutination inhibition test (HAI).

                  TABLE 7                                                         ______________________________________                                        ANTIBODY RESPONSE (HAI)                                                       EQUINE INFLUENZA A2                                                           Horse No.  Pre    1 week    2 weeks                                                                              4 weeks                                    ______________________________________                                        19         <8     128       256    128                                        23          8      64       128    128                                        61         <8     128       256    128                                        ______________________________________                                    

Once again, the antibody titers obtained are greater than required forprotection.

It should be noted that this disclosure is concerned with both a novelinfluenza culture system and the use of the medium to produce a novelinfluenza vaccine. The liquid cell culture system used in this inventionentains the use of susceptible cells, influenza viruses, a nutrientmedium and a proteolytic enzyme, but unlike past systems (e.g. theTobita et al. reference), does not require the use of agar which reducesthe system to a semi-solid state. The exclusion of the agar thus enableslarge scale production of viruses in the conventional manner known tothe art and results in the production of viral fluids of sufficientlyhigh titers for the preparation of vaccines.

The influenza vaccine preparation itself comprises effective amounts ofone or more strains of given influenza virus particles and apharmaceutically acceptable carrier, the total preparation, preferablyin aqueous form, being substantially free of reactive proteins such asegg proteins. As used herein, the term substantially free of egg proteinmeans that the only possible source of egg protein in the vaccinepreparations is the seed virus which is diluted >1:100,000.

The vaccines are administered to animals by various routes, includingintramuscular, intravenous, subcutaneous, intratracheal, intranasal, orby aerosol spray and the vaccines are contemplated for beneficial use ina variety of animals, including human, equine, porcine, and aviangroups.

The viral preparations produced by this invention may be diluted withwater to adjust their potency, and they may have added to themstabilizers such as sucrose, dextrose, lactose, or other non-toxicsubstances. The viral preparations may be desiccated by freeze dryingfor storage purposes or for subsequent formulation into attenuatedvaccines or they may be chemically inactivated for the preparation ofkilled virus vaccines.

It can be appreciated that all of the virus strains of the aboveExamples fall into the Genus Influenza virus within the OrthomyxoviridaeClassification. See, for example, Principles of Animal Virology, W. K.Joklik, Appleton-Century-Crofts/ 1980, pages 52 and 53. Accordingly, itis intended that this disclosure should enable one skilled in the art touse the above-described methods to prepare high-titered (e.g. EID₅₀ /mlof at least about 10⁷) vaccines for any virus of the Orthomyxoviridaeclass. Regarding the tissue culture cell use, it is only necessary thatthe cell be susceptible to the specific virus strain of the GenusInfluenza from which a vaccine is to be prepared. It is thought to bewell within the skill of a component microbiologist or virologist toselect such a susceptible cell vis-a-vis a given virus strain of theOrthomyxoviridae class. In a typical situation a virologist would selectthe best growing cells from known cell stocks, infect them with thevirus strain in the presence of protease, and observe for production ofcytopathic effect. Fluids from all infected cells producing cytopathiceffects would be titrated for virus concentration. Experiments in ourlaboratory indicated that many cell types including Bovine Kidney. Vero,and Canine Kidney support growth of Influenza virus when using theprocedure described herein.

As described above, an essential feature of the present invention isthat the incubation (virus replication) step be in the presence of aprotease under conditions sufficient to overcome the one-step growthcycle of the virus. As can be seen from the data, and especially asshown by the Figures, when a protease (such as trypsin) is included (inthe range of about 4-25 micrograms/ml culture medium) in the culture, anEID₅₀ /ml titer of at least about 10⁷ is obtained. Obtaining such a hightiter (in many cases ≧10⁹ EID₅₀ /ml) now provides a means foreconomically making Influenza Virus vaccines without the use of costlyembryonated eggs. It should be pointed out that the surprisingly hightiter results shown in the Tables and Figures are due solely to thepresence of the indicated amounts of the protease (trypsin) in theinfected tissue culture. In cases where trypsinization techniques areused (e.g. in removing cell layers from a surface, using significantlylarger amounts of the enzyme, 0.5 to 1.0 mg/ml) it is common practice,prior to virus infection, to remove or inactivate the enzyme to avoiddigestion of the cells. Removal of the trypsin is accomplished by simplycentrifuging the trypsin/cell mixture and pouring off the supernatanttrypsin solution or by simply diluting the active trypsin out of thesystem. Alternatively, any remaining active trypsin may be neutralized(inactivated) by assuring the presence of known trypsin activityneutralizers (i.e. the presence of serum, used for nutrative purposes inthe cell culture, will assure the inactivation of trypsin and precludethe carry over of active trypsin to the virus-infected cell culture). Ineither case (trypsin removal or inactivation), it is important to notethat the pre-infection liquid cell culture of this disclosure issubstantially free of proteolytic activity. As used herein, theexpression substantially free of proteolytic activity means thatwhatever activity may remain it is not sufficient to overcome singlestep growth cycle. Additional fresh trypsin may be added to accomplishthe virus replication required by the invention disclosed herein. Evenif there were some residual trypsin which somehow escaped removal orinactivation techniques such residual amounts would correspond to the"zero" amounts in the Figures (the controls) since those cells had beentransferred using conventional trypsinizing technique and, hence, not bepresent in quantities sufficient to lead to the high titer results.Because of the use of the protease during the replication step (incontrast to any prior trypsination steps which would eliminate any suchprotease activity), the final vaccine product of this invention willcontain measurable amounts of protease activity. In the case of trypsinuse, the final Influenza virus vaccine will typically contain at least 1to 10 units of trypsin activity per milliliter of Vaccine preparation(e.g. vaccine in an aqueous carried), a feature not associated withknown Influenza virus vaccines. One unit is defined as the ΔA₂₅₃ of0.001/min. with N alpha-benyl-L-arginine ethylester (BAEE). This featurehelps distinguish the vaccine of this invention from those of the priorart. Thus, the typical vaccine of this invention contains virus grown toat least 10⁷ EID₅₀ /ml, a trypsin activity of at least about 1 to 10units per mil, and includes a pharmaceutically acceptable liquid carrierwhich is relatively clear, non-cloudy, and essentially free of eggprotein. As used herein, the expression pharmaceutically acceptableliquid carrier includes a tissue culture fluid which consists of a basalsalts solution, amino acids, vitamins, other growth nutrients,antibiotics and a fungistat.

It should be understood that the above examples are merely illustrativeand that the scope of this disclosure should be limited only by thefollowing claims.

We claim:
 1. A method of preparing an influenza virus vaccine whichcomprises the steps of:(a) infecting a portion of the cells of a liquidcell culture substantially free of proteolytic enzyme with an influenzavirus; (b) incubating the infected culture in the presence of aproteolytic enzyme under conditions sufficient to assure furtherinfection and replication of the virus in cells other than theoriginally infected cells and an EID₅₀ /ml titer of at least about 10⁷ ;(c) harvesting the virus from the culture, and; (d) preparing a vaccinefrom the harvested virus.
 2. The method of claim 1 wherein the cellculture of step (a) comprises a confluent monolayer of cells.
 3. Themethod of claim 1 wherein the cell culture of step (a) comprises aliquid suspension of cells.
 4. The method of claim 1 wherein the vaccinepreparation of step (d) includes the step of inactivating the harvestedvirus.
 5. The method of claim 1 wherein the vaccine preparation of step(d) includes the step of attenuating the harvested virus.
 6. The methodof claim 1 wherein the proteolytic enzyme of step (b) is selected fromtrypsin, chymotrypsin, pepsin, pancreatin, papain, pronase andcarboxypeptidase.
 7. The method of claim 1 wherein the virus is anequine influenza virus.
 8. The method of claim 7 wherein the virus is astrain selected from equine influenza A1 and equine influenza A2 virusstrains.
 9. The method of claim 1 wherein the virus is a human influenzavirus.
 10. The method of claim 9 wherein the virus is selected fromB/Hong Kong, A/Texas, and A/USSR virus strains.
 11. The method of claim1 wherein the cells of the cell culture are dog kidney cells.
 12. Themethod of claim 1 wherein the cell culture of step (a) is a dog kidneycell culture, the virus of step (a) is an equine influenza virus, andthe enzyme of step (b) is trypsin.
 13. The method of claim 1 wherein thecell culture of step (a) is a dog kidney cell culture, the virus of step(a) is a human influenza virus, and the enzyme of step (b) is trypsin.14. The method of claim 1 wherein the enzyme of step (b) is present inan amount sufficient to assure infectivity and replication of the virusin culture cells other than the cells comprising that portion infectedin step (a).
 15. The method of claim 14 wherein the amount of trypsinranges from about 4 to about 25 micrograms per ml of culture media..Iadd.
 16. A method of preparing an influenza virus vaccine whichcomprises the steps of(a) infecting a portion of the cells of a liquidcell culture substantially free of proteolytic enzyme with an influenzavirus; (b) incubating the already infected culture in the presence of aproteolytic enzyme present in an amount sufficient to assure furtherinfection and replication of the virus in cells other than theoriginally infected cells until the EID₅₀ /ml titer has increased by afactor of at least about 10² ; (c) harvesting the virus from theculture; and (d) preparing a vaccine from the harvested virus. .Iaddend..Iadd.
 17. The method of claim 16 wherein the proteolytic enzyme ispresent at a concentration of between about 4 and 25 micrograms per mlof culture medium. .Iaddend. .Iadd.18. The method of claim 16 whereinthe cells are incubated with the infecting inoculum for at least aboutone hour before the proteolytic enzyme is added. .Iaddend. .Iadd.19. Themethod of claim 16 wherein the proteolytic enzyme is selected from thegroup consisting of trypsin, chymotrypsin, pepsin, pancreatin, pronaseand carboxypeptidase. .Iaddend. .Iadd.20. The method of claim 19 whereinthe proteolytic enzyme is trypsin. .Iaddend. .Iadd.21. The method ofclaim 20 wherein the trypsin is present at a concentration of betweenabout 4 and 25 micrograms per ml of culture medium. .Iaddend. .Iadd.22.The method of claim 21 wherein the cells are incubated with theinfecting inoculum for at least about one hour before the trypsin isadded. .Iaddend. .Iadd.23. The method of claim 22 wherein the virus isan equine influenza virus. .Iaddend. .Iadd.24. The method of claim 23wherein the virus is a strain selected from the group consisting of theA1 and A2 equine influenza strains. .Iaddend. .Iadd.25. The method ofclaim 22 wherein the virus is a human influenza virus. .Iaddend..Iadd.26. The method of claim 25 wherein the virus is selected from thegroup consisting of the B/Hong Kong, A/Texas, and A/USSR human influenzastrains. .Iaddend. .Iadd.27. The method of claim 23 or claim 24 or claim25 or claim 26 wherein the cells of the cell culture are dog kidneycells. .Iaddend. .Iadd.28. The method of claim 16 wherein the cellculture which is infected is a liquid cell suspension culture. .Iaddend..Iadd.29. The method of claim 28 wherein the cell culture is maintainedas a liquid cell suspension culture until the virus is harvested..Iaddend. .Iadd.30. The method of claim 16 wherein the cell culture isincubated with the proteolytic enzyme as a confluent monolayer of cells..Iaddend. .Iadd.31. The method of claim 30 wherein the cell culture isincubated with the proteolytic enzyme in a roller bottle, Roux bottle orPovitsky flask. .Iaddend. .Iadd.32. The method of claim 31 wherein thecell culture is incubated with the proteolytic enzyme in a rollerbottle. .Iaddend. .Iadd.33. A method of preparing an equine influenzavaccine which is essentially free of egg proteins which comprises thesteps of(a) incubating a liquid cell culture with an amount of aninoculum of the influenza virus sufficient to infect a portion but notall of the cells in the absence of any substantial amount of proteolyticenzyme for between about 1 to 72 hours; (b) combining the infected cellculture with between about 4 and 25 micrograms per ml of culture oftrypsin and incubating until maximum cytopathic effect is observed andthe EID₅₀ /ml titer has increased by a factor of at least about 10² ;(c) harvesting the virus from the culture; and (d) preparing a vaccinefrom the culture. .Iaddend. .Iadd.34. The method of claim 33 wherein thecells of the culture are dog kidney cells. .Iaddend.